Figure 3.

Acetate produced from gut microbiota may induce the dysregulation of cholesterol homeostasis. HK-2 cells were incubated with or without acetate (10 mM) in the presence or absence of glucose (30 mM) for 24 h. (A) Linear discriminant analysis (LDA) effect size (LEfSe) algorithm of OUT tables was used to determine taxa that best characterize each biological class in faeces of rats (n=6). (B) The serum acetate level of rats was measured by gas chromatographic analysis (n=4-6). ^**P<0.01 versus the control, ^###P<0.01 versus DM. (C) Correlation analysis between the serum acetate level and cholesterol content of kidneys was determined by Spearman's R coefficient (n=14). (D) Oil red O staining (scale bar, 200 μm, original magnification ×400) and Filipin staining (scale bar, 100 μm, original magnification ×200) were used to observe lipid accumulation in HK-2 cells. (E) Cholesterol content in HK-2 cells was quantified by free cholesterol quantification assays. (n=3 independent experiments). ^*P<0.05 versus the control. (F) Protein expression of HMGR, LDLr, CD36, and CXCL16 in HK-2 cells were measured by immunofluorescence staining (scale bar, 20 μm, original magnification ×800) and (G and H) Western Blotting (n=3-6 independent experiments). ^*P<0.05 versus the control, ^**P<0.01 versus the control, ^***P<0.001 versus the control. (I-J) Protein expression of TGF-β1 and CTGF was measured by Western Blotting (n=5 independent experiments). *P<0.05 versus the control, **P<0.01 versus the control. The density of protein bands was quantified by software ImageJ, which was normalized by comparison with β-actin and expressed as the percentage of control. Data are expressed as the mean ± SD. HMGR, 3-hydroxy-3-methylglutaryl coenzyme A reductase. LDLr, low-density lipoprotein, receptor. CXCL16, CXC chemokine ligand 16. TGF-β1, transforming growth factor-β1. CTGF, connective tissue growth factor.